Antigenic chimeric viruses in which the structural genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding genes of wild-type rDEN4 or vaccine candidate rDEN4delta30 which contains a 30 nucleotide deletion in the 3-UTR (untranslated region). The two DEN2/4 chimeric viruses lacking the delta30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions leads to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the delta30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the delta30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the delta30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the delta30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4delta30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials. An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1,248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and it contained a single coding mutation: a C to U mutation at nucleotide 7129 resulting in a Pro to Leu change in amino acid 101 of the non-structural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (delta30) decreased the ability of the double mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host. Mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. We have identified eight missense mutations in dengue virus type 4 (DEN4) that increase the plaque size and kinetics of replication of recombinant DEN4 (rDEN4) virus in Vero cells. rDEN4 viruses bearing these Vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice. Two recombinant viruses expressing distinct mutations in NS3 were both restricted in replication in the brains of suckling mice. In contrast, six recombinant viruses, each encoding individual mutations in NS4B (five) or in NS5 (one), were not attenuated in mouse brain. Recombinant viruses encoding various combinations of these Vero cell adaptation mutations did not demonstrate enhanced replication in Vero cells over that exhibited by the single mutations. Finally, addition of a subset of the above non-attenuating, adaptation mutations to a DEN2/4 chimeric vaccine candidate was found to increase the virus yield in Vero cells by up to 500-fold.